Thyroid hormone (TH) is a critical regulator of metabolism and body weight in humans. Indeed, TH has been shown to regulate energy expenditure, lipid levels and insulin sensitivity to improve metabolic outcome. Because of these beneficial effects, TH or one of its analogs is a potential therapeutic for a wide variety of metabolic diseases. However, if excessively dosed TH has significant side effects especially on the heart and bone. Remarkably, TH action in humans is determined almost exclusively by the use of the thyrotropin (TSH) assay which depends only upon TH action at the level of the hypothalamus and pituitary. Thus, there is a critical need for improved biomarkers that would allow for tailoring of TH therapy to improve metabolic health and allow for enhanced safety. To begin to accomplish this we have used metabolomic profiling and mouse genetic models to begin to characterize TH sensitive pathways in vivo. Our preliminary work has identified unique metabolites that are regulated by TH that serve both as biomarkers and also as mediators of thyroid hormone action. In this proposal in the first aim we will extend our analysis of the regulation of metabolite profiles by TH in a tissue-specific and TH receptor specific fashion and extend our analysis into humans for the first time. In the second Aim we will determine the mechanism by which TH regulates methionine metabolism which is likely critical to TH ability to control gene expression and regulate programs such as fatty acid oxidation. In the third Aim we will use novel genetic mouse models to determine how TH controls cholesterol metabolism gaining further insight into a key pathway regulated by TH. Together, completion of these Aims will provide for a new platform for both understanding and defining TH action in vivo.